Blasticidin-s addition products and their manufacturing process



M y 11', 1965 YUSUKE SUMIKI ETAL 3,183,153

BLASTICIDIN-S ADDITION PRODUCTS AND THEIR MANUFACTURING PROCESS FiledJune 5, 1961 I 2 Sheets-Sheer. 1

F/EE'Z WAVENUMBER (cm-9 s uoudaossv NOIldHOSQV BLASTICIDIN-S ADDITIONPRODUCTS AND THEIR MANUFACTURING PROCESS Filed June 5. 1961 May 11, 1965YUSUKE SUMIKI ETAL 2 Sheets-Sheet 2 QQR 7 com NOIicIHOSSV 3 NOlidHOQSVUnited States Patent 3,183,153 BLASTICIDIN-S ADDITION PRODUCTS AND THEIRMANUFACTURING PROCESS Yusuke Sumiki, Hamao Umezawa, K azuo Fukunaga,Hiroshi Yonehara and Hideo Kubo, Tokyo, Shigeo Fujita, Kanagawa-ku,Yokohama-shi, Kanagawa-ken, Shiro Shirato and Kotaro Takaga, Tokyo,Masaji Kato, Ashigara-kami-gun, Kanagawa-ken, and Shinichiro Esumi andTadao Ono, Tokyo, Japan, assignors to Zaidan-Hojin Nihon Kosei BusshitsuGakujyutsu Kyogikai and Kaken Kagaku Kabushiki Kaisha, Tokyo, Japan, acorporation of Japan Filed June 5, 1961, Ser. No. 114,895 Claimspriority, application Japan, June 9, 1960,

2 Claims. (Cl. 167-65) The present invention relates to a process forthe production of agricultural chemicals including as a principalconstituent Blasticidin-S, an antibiotic substance againstphytopathogenic fungi. More characteristically speaking, it is a processwherein the Blasticidin-S, producing strain, such as Streptomycesgriseochromogenes Fukunaga (ATCC No. 14511) or Streptomyces albus var.pathocidicus (ATCC No. 14510) is cultivated at an appropriate conditionand, thereafter, precipitants for basic substances are added directly tothe resulting culture broth filtrate or to extracts from methacrylicacid type cation exchange resins.

Blasticidin-S (abbreviated as B-S in the following) is originally theantifungal and antibacterial substance which has first been isolated incrystalline form by Yusuke Sumiki, Hiroshi Yonehara et al., and whichhas been reported (in the Journal of Antibiotics, Ser. A XI, No. 1,1958) of its remarkable autibotic property, especially againstPiricularia aryzae, Pseudomonas species, showing the characteristicproperties as described later.

Blasticidin-S producing strains are reported also as follows:

Streptomyces griseochromogenes Fukunaga (the Journal of Antibotics, Ser.A, vol. Xl, No. 1, January 1958), and Streptomyces albus var.pathocia'icus (presented at the meeting of Nippon Nogei Kagaku Kai (i.e.the Agricultural Chemistry Society of Japan) held April 1961 by J.Nagatsu, K. Anzai and Y. Surniki).

Blasticidin-S free base is a white needle crystal, melt ing at 235 to236 C. under decomposition. The stability of 3-8 in aqueous solution atVarying pH values was examined at 100 C. and found that it-shows thehighest stability at pH 5.0 to 7.0 and less stability at pH 4.0 ascompared, with cases of pH 2.0 and pH 8 to 9. The free base ofBlasticidin-S is soluble in water and acetic acid, but insoluble inmethanol, ethanol, acetone, benzene, ether, ethyl acetate, butylacetate, chloroform, carbon tetrachloride, methyl ethyl ketone,cyclohexane, xylene, tetrahydrofuran, methyl isobutyl ketone, pyridineand dioxane. It is negative in ferric chloride, Fehling, Tollens, sodiumnitroprusside, triphenyl tetrazonium chloride, bromonitroso, maltol,millon, Ehrlich, Sakaguchi, Moliseh, biuret, xanthroprotein and Grafsketone, but

positive in diazo, 2,4-dinitrophenylhydrazine, Grafs aldehyde,ammoniacal silver nitrate and ninhydrin.

Decomposition point of Blasticidin-S hydrochloride is 224 to 225 C. Thatof the picrate is 200 to 202 C., and

3,183,153 Patented May 11, 1955 the helianthate is 224 to 225 C. Opticalrotation of the free base [a] =+108.4 (c.=1% in Water). The measuredvalue of the molecular weight of Blasticidin-S were 356.36 by titrationequivalent with perchloric acid, 355.8 by the analysis of the doublesalt with platinum chloride and 300 to 450 by Berger-Akiya method.

Elementary analysis of Blasticidin-S-Calc. c,.,H ,0,N (M.W. 352): c,47.22; N, 23.85. C, 47.11; H, 5.83; N, 24.46. a

The ultraviolet absorption spectrum absorption maximum is for Eit... 349at 275 m,

in N/ 10 hydrochloric acid and in case of N/ 10 sodium hydroxide itsvalue is Infrared absorption bands are observed in the following wavenumbers: 2318, 3130, 2860, 1675, 1614, 1600, 1557, 1492, 142-2, 1400, 1353, 1300, 1282, 1235, 1200, 111:1, 1067, 1042,1002, 943, 860, 822, 775,714 cm- Quantitative determination of BS: Biological assay method due toBacillus cereus IAM 1729 is used. As a standard for activity, a highpurity Blasticidin-S crystals (1000 u./mg.) are used.

Biological activities of Blasticidin-S were examined by the agar streakdilution method and the broth dilution method, the results are showninTable I.

TABLE I.ANTIBIOTIC SPECTRUM Test organisms: Growth inhibition, meg/ml.

Bacillus subtilis 50 Bacillus lactis Escherichia coli 50 Pseudomonasfluorescens 5 Mycobacterium tuberculosis ATCC 607 50 Penicillium notatum100 Penicillium chrysogenum Q 176 100 Aspergillus oryzae 100 Candidaalbricans 100 Saccharomyces cerevisiae 100 Torula utilis 100 Piriculariaoryzae 5-10 Sclerotinia mali l0 Sclerotinia araclidis 100 Bacteriumcitri 5 Bacterium aroideae 5 Pseudomonas tabaci S Xanthomonas citri 50Alternaria kikuchiana 50 Found:

The primary object of this invention is to provide the new B-Spreparations which show less toxicity and high effectiveness asagricultural chemical. The usual methods adopted for the manufacture of3-3 preparations are not suitable for the manufacture of agriculturalchemicals, which should be low cost, since those methods are complicated and on a laboratory scale as described in the above-citedliteratures.

A second object of the present invention is to produce B-S preparationsat high yields and in conditions especially advantageous for massproduction. In order to accomplish the first object referred to above,the present inventors have conducted investigation on' the followingchemicals shown in Table II for the purpose of making above-mentionedB-S preparations slightly soluble in water, without reducing therapeuticeffects of agricultural chemicals. From the result of investigationsreferred to above, it has been realized that B-S preparations andslightly soluble powders or crystalline addition compounds can beprocessed at high yield, which have been a selected as an agriculturalchemical having excellent effects.

TABLE IV Purity, Solubility in B-S add. product uJmg. water (u./ml.)

Blastleidin-S 1, 000 20, 000 B8 dodeeylbenzene sultonate. 580 105 B-Slaurylsulfate 630 235 13-8 benzylaminophenylsulfonate 595 1, 380

NE. BS in the filtrate was determined after dissolved in distilled waterat C. under shaking for 2 hours.

TABLE II Quantity of Appearance of Purity in 13-8 Preoipltates for basicsubstances precipitation precipitation potency; units/mg.

Sodium dodecylbenzene sulfonate Powdery- 457 Sodium dibutylnap'nthalenesulfonate. 501 Sodium tetradecyl sulfonate 517 'Iergitol 480Cresolsulfonic acid-formalin condensate. 357 Castor oil, sulfate 3Sodium diallzylbenzene sulfonata. 346 Sodium laurylsesqniphosphate. 330Sodium lauryl sulfate 4 2 Dialkyl sulfosuccinate 99 Calciumdinaphthylmethan disulfonate. 354 Sodium naphthalene sulfonate 346Higher fatty acid-Inethyltaurin con- 336 donsate. Sodium cetyloleylsulfate 406 Sodium benzylamiuophenyl sulfonate 670 Sodium dipropylbenzylaminophenyl 529 sulfonate. Sodium picrate. 495

Sodium flariamatev++-l- .do 366 Methyl orange- Yellow orange 384 powder.Ammonium relneckato. Red purple 350 powder. Sodium picrolonate Yellow412 powder. Sodium pentaehlorophenolate White powder- 400 Sodiumpatatoluol sulfonate. Pasty 422 Sodium fl-naphthalene sulionate Whitepowder: 4.50 sodium 2,-1dinitro-naphtl1al 7-su lfonate Yellow 430 powder1 Sodium tetradeeyl sulfate.

The results of experiments on precipitation due to various precipitantsfor basic substances are respectively shown in Table II.

Decomposition points and results of elementary analyses on severalexamples of Blastz'cz'din addition products Colorless needle crystallineB-S addition products can be separated by procedure as follows: B-S isreacted with sodium lauryl sulfate or sodium benzylamino-phenylsulfonate in heated (about C.) water solution and then the additionproduct is crystallized by cooling.

are shown in Table III. so The amount of addition is from equivalent todouble TABLE III Decomposi- Analyses, percent tion goint,

' Carbon Hydrogen Nitrogen Sulfur Blasticidin-S (Id-S) 235-236 47. 11 5.83 24. .6 0 13-5 lauryl-sulfate 239-240 51. 13 7. 15. 61 4. 56 13-8dodeeyl-benzene sulfonate -a 241-242 55. 46 7.72 14. 87 4. 17 13-5benzylamino-phonylsulfouate. 235-238 52. 7G 5. 84 16. 87 4. 44 13-8picrate. 200-202 41. 34 4. 18 22. 24- B-S heliauthate 224-225 49. 25 5.93 20. 47 2. 52

the content of B-S to accomplishing the precipitation of the additionproducts referred to above. When however, a large quantity of impuritiesof organic basic substances coexists, it is necessary to add the sulfateor the sulfonate somewhat in excess. 7

B-S addition products obtained by the investigation con-ducted by thepresent inventors have been found to be less toxic to rat (LD than B-Sas apparent from and of practical advantages as an agriculturalchemical. 10

The results of oral toxicity tests listed in Table V.

In order to accomplish the second object of this invention referred toabove, first based on the result of investigations with respect to theenhancement of 13-8 productivity or high potency of the culture media offermentation .of 13-8 producing strain, good results were obtainedparticularly by the addition of embryo of corn, bran, or yeast, as thecomponents of culture media with usually employed ingredients such' assugars, soybean powder, and inorganic salts. One example of the resultsof the experiments is shown in Table VII.

TABLE V B-S precipl- Antiiungal Toxic to Acid Surface-active agent tatedper mg. value, rat LDio, No. of surfaceuJmg. mgJkg.

active agent, mg.

Sodium alkyl benzene sulfonate..- 0 87 426 57 a Sodium laurylsulfonate 1. 18 470 39. 5 Sodium cetyl oleyl sulfate 0. 77 415 46. 2Cresol sulfonic acidformalin con- 0 47 381 38. O

densate. Sodium lauryl cesquiphosphate--. 0. 63 341 38. 5 Blastieidin-Scrystal 1, 000 16. 3

In addition, it has been recognized that since drug penetrates slowlyinto plant the durability of chemicals increases and further thetoxicity against plant cells de- 40 creases remarkably. Particularly,B-S addition products with surface active'agents have good spreadingproperty to plant surface and consequently the .efiiciency ofagricultural drugs, increases to show advantageous exceedingly inpractical use.

The results of the therapeutically examinations of B-S against thePiricularia oryzae-infections are as shown in Table VI. Obvioussuperiority has been observed as compared with therapeutic effects ofmercury preparation-s usually used.

Test c0nditi0ns.-Rice plant cultivatedin green house: average growth,20-30 cm.; the amount of chemicals dusted, mL/pot on a turntable bymeans of a lb. spray gun; the atmospheric temperature at the time ofTABLE VH [Basal medium: Sugar 4%, soybean powder 3%, table salt 0.6%]

' Addition to basal medium, embryo of- B-S eoncen- Ratio,

tration, percent meg/ml. Wheat Bran Yeast In the next place, as aprocess of isolating B-S, a process for the purification by theextraction due to ordinary active carbon and iron exchange resin,accompanied by alumina chromatography has been adopted. The result ofexperiments on the condition of simple purification of B4; from culturebroth is shown in Table dusting, 205 0. VIII.

TABLE VI Ooncen- Total number Number Thera- Specimen tration, of spotson the of acute Ratio peutie p.p.rn. infected leaves spots B/A value,

A percent 1. 13-8 laugyl siilfate 10 154 12 7. 8 91. 2 2. 13-8 do ecenzene sulionatey 10 228 45 19. 7 77. 9 B-S ben a sulionate z 10 262 176.5 92.7 4. Phenyl mercuric acetate- 20 252 141 5G. 0 48. 4 5. Nottreated 399 356 89.0

TABLE VIII Adsorption 0.5N-Hl Resin Form amount, elution Yield,

mgJR-H n11. rate in mg./R-H gr. percent Methacrylic acid type H 109. 076.0 83, 0

RC OOH (Amberlite IRCO) Na 123.0 19.6 24. 5

Salicylic acid type H 58. 8 80. 4 47. 2

COOH (Kaken resin) Na 51. 0 91. 8 4G. 8

Sulfonic acid type H 82. 0 4 4 12. 3

/SOaH (Duolitc Cl0) Na 99.0 4 15 4 14. 8

Phenol type H H 17.0 82.1 14. 0 (Duolite 8-30) Na 21. 4 69. 5 10. 9

1 Rohm & Haas 00. product.

2 Kakcn Chemical 00. product. 8 Chemical Process 00. product. 4 Maximum.

As apparent in the above-shown table, salicylic acid type and phenoltype resins possess relatively good elution power, whereas theadsorption quantity of those resins are inferior to metacrylic acidtype, and sulfonic acid type resin shows remarkabl poor elution rate indilute acid. In the present invention, the optimum pH with respect toadsorption is appropriate at 5 to 7 in view of stability of B S. Incarrying out this invention, it is preferable to preliminarily convertresin in H-form with sulfuric or hydrochloric acid then wash thoroughlywith water and make the culture filtrate in the neighbourhood ofneutrality. In this case, resins whose pH has been adjusted beforehandwith phosphate buifer solution of pH 7 of H-form resin, about 50% ofwhich has been changed to Na-form resin show no significant differencesin adsorption from the former. While Na-form resins simply washed inwater are remarkably bad in adsorption. As referred to above in thepresent invention, the condition of elution from BS adsorbed resins ispreferable to use 1.0-0.1 N-dilute acid, but in industry sulfuric acidOr hydrochloric acid is used generally and preferably.

According to the present invention, as referred to above, low toxicityand excellent agricultural chemicals can be produced economically andadvantageously, by enhancing yields due to the improvement ofproductivity of B-S during the cultivation process and by raisingefficiency in the extraction process and simultaneously by making theproduct to be B-S additives slightly soluble in water.

Example 1 Micro-organisms of Streptomyces griseochromogenes Fukunaga,i.e. the producing strain for Blasticidin-S, are inoculated in a mediumcomprising 2% glucose, 1% peptone, 1% meat extract and 0.2% common salt,and cultured under agitation for about 48 hours. The culture justreferred to is designated as a preliminary culture. The resultingprecultured substance is then inoculated in a medium comprising 5%sugar, 3% defatted' soy-bean powders, 2% embryo of wheat and 0.6% commonsalt, which has been prepared in a 600 litre fermentation tank, andcultured for approximately 24 hours at 29 C. The culture referred tofollowing to the preliminaryc'ulture is designated as a secondpreliminary culture. The resulting substance is grafted in theabove-mentioned medium contained in a 600 litre fermentation tank andsubjected then to fermentation at 29 C. under agitation with aeration.Blasticidin-S is produced in 120 hours of fermentation, when the amountof production reaches maximum and the potency in the liquid arrives at5700 mcg./ ml. The fermented liquid is added with diatomaceous earth asfilter aid and filtered and 4 kilolitres of transparent filtratesolution is adsorbed at 20 space velocity on 500 litres by volume inwater of methacrylic acid type cation exchange resin amberlite TRC50 H,and dissolved out with 0.5 N hydrochloric acid at fiow rate of spacevelocity 0.3.

In 500 litres, eluate B-S contains 36.4 mg./ml., total activity of B-S:18.2 kg., and its extraction yield showed The crude BS hydrochlorideaqueous solution is adjusted at pH 6, heated at 40 C. and added withaqueous solution of 20 kg. sodium lauryl sulfate dissolved in 400 litresWarm water, reacted under stirring and stood over night at cool place.Then crystals precipitated are separated and 40 kg. of dry crystal isobtained. The purity is 450 u./mg. and 3-8 preparations for Pir. oryzaeis prepared from this substance after addition with suitableingredients.

Example 2 Starting from the Blasticidin-S cultured broth, 5 litres crudeB-S sulfate aqueous solution in 15 mg./rnl. B-S concentration, extractedwith methacrylic acid type cation exchange resin: Duorite CS-l01, isadjusted at around pH 6, heated at 40 to 50 C. and added with aqueoussolution of gr. sodium alkylbenzenesulfonate dissolved in 1700 ml.water, vigorously stirred and stood in an ice chamber overnight; thenamorphous precipitates formed are separated by filtration.

, The dry matter yields at gr. (B-S purity: 435 u./mg.).

Example 3 The yield as dry matter of BS additive: 75 gr.; purity:

340 u./ mg.

' 9 Example 4 15 litres crude aqueous solution containing 1.0 kg.Blasticidin-S hydrochloride is added with aqueous solution of 4 kg.sodium benzylaminophenyl sulfonate dissolved in 15 litres warm water,mixed and stood still over-night and filtered.

Unit in mother liquor: 120 u./ml.; t-otal precipitated dry matter: 1.9kg.; purity 430 u./ml.; yield: 94%. What We claim is:

1. The process for preparing Blasticidin-S addition salts comprisingcontacting a Blasticidin-S containing broth with a methacrylic acidcation-exchange resin preadjusted at pH 5-7, to absorb Blasticidin-S onsaid resin, eluting said resin with 0.1 to 1.0 N dilute acid, thenadding to the resulting eluate a surface-active agent containing ananion radical selected from the group consisting of sulfonate andsulfate to produce a difficulty water-soluble addition salt of saidBlasticidin-S, and recovering the addition salt product.

, 10 2. The Blasticidin-S addition salt produced by the process of claim1.

References Cited by the Examiner UNITED STATES PATENTS 2,528,188 10/50Taylor 260-210 2,537,934

OTHER REFERENCES JULIAN S. LEVITT, Primary Examiner.

MORRIS O. WOLK, IRVING MARCUS, LEWIS GO'ITS, Examiners.

1 /51 Lott 260-210

1. THE PROCESS FOR PREPARING BLASTICIDIN-S ADDITION SALTS COMPRISINGCONTACTING A BLASTICIDIN-S CONTAINING BROTH WITH A METHACRYLIC ACIDCATION-EXCHANGE RESIN PREADJUSTED AT PH 5-7, TO ABSORB BLASTICIDIN-S ONSAID RESIN, ELUTING SAID RESIN WITH 0.1 TO 1.0 N DILUTE ACID, THENADDING TO THE RESULTING ELUATE A SURFACE-ACTIVE AGENT CONTAINING ANANION RADICAL SELECTED FROM THE GROUP CONSISTING OF SULFONATE ANDSULFATE TO PRODUCE A DIFFICULTY WATER-SOLUBLE ADDITION SALT OF SAIDBLASTICIDIN-S, AND RECOVERING THE ADDITION SALT PRODUCT.